The present invention is concerned with a genetically manipulated infectious replicating non-segmented negative-stranded RNA virus mutant and a process for the preparation of such a mutant.
Rabies virus (RV) is an example of a non-segmented negative-stranded RNA virus of the Rhabdoviridae family. Other species belonging to this family are vesicular stomatitis virus (VSV), infectious hematopoietic necrosis virus (IHNV) viral haemorrhagic septicaemia virus (VHS, Egtved virus), bovine ephemeral fever virus (BEFV), and sonchus yellow net virus (SYNV).
Beside the family of Rhabdoviridae also viruses belonging to the Paramyxoviridae (e.g. sendai virus (SV), para-influenza virus (PIV) type 2 and 3, Newcastle disease virus (NDV), mumps virus (MUV), measles virus (MEV) and canine distemper virus (CDV)) and Filoviridae, and several viruses not assigned to a family (e.g. Borna disease virus; BDV) have a nonsegmented negative-stranded RNA genome.
The overall genomic organisation in the non-segmented negative-stranded RNA viruses of the various families is comparable. Especially between the paramyxoviridae and the rhabdoviridae, there are only minor differences in the overall genomic organisation (Tordo et al., Seminars in Virology 3: 341-357, 1992).
RV can infect all warm-blooded animals, and in nearly all instances after establishment of symptoms the infection ends in death. Dog rabies is still important in many parts of the world: infected dogs cause most of the estimated 75,000 human rabies cases that occur each year world-wide. In many countries of Europe, and in the United States and Canada, wildlife rabies has been increasing in importance.
The clinical features of rabies are similar in most species, but there is great variation between individuals. Following the bite of a rabid animal the incubation period is usually between 14 and 90 days, but may be considerably longer, and incubation periods of over a year have been documented. Two clinical forms of the disease are recognized furious and dumb or paralytic. In the furious form, the animal becomes restless, nervous, aggressive, and often dangerous as it loses all fear of humans and bites at anything that gains its attention. The animal often cannot swallow, giving rise to the synonym for the disease, "hydrophobia". There is often excessive salivation, exaggerated responses to light and sound, and hyperesthesia. As the encephalitis progresses, fury gives way to paralysis, and the animal manifests the same clinical features as seen throughout in the dumb form of the disease. Terminally, there are often convulsive seizures, coma, and respiratory arrest, with death occurring 2-7 days after the onset of clinical signs.
Rabies virus enters the body in the bite or occasionally the scratch of a rabid animal, or when virus-loaded saliva from a rabid animal enters an open wound. Viral replication in the bite site, in muscle, is followed by invasion of peripheral nerve endings and central movement of viral genome in the cytoplasm of axons to the central nervous system. Viral entry into the spinal cord and then the brain (particularly the limbic system) is associated with clinical signs of neuronal dysfunction. Usually, at about the same time that central nervous system infection causes fury, virions are also shed from the apical end of mucus-secreting cells in the salivary glands and are delivered in high concentrations into saliva.
Throughout the course of rabies, host inflammatory and specific immune responses are only minimally stimulated; the most likely reasons for this are because the infection is noncytopathic in muscle and in nerve cells and because the infection is largely concentrated in the immunologically sequestered environment of the nervous system.
RV virions like all Rhabdoviruses are composed of two major structural components: a nucleocapsid or ribonucleoprotein (RNP) core and an envelope in the form of a bilayer membrane surrounding the RNP core. The infectious component of all Rhabdoviruses is the RNP core. The genomic RNA is of negative sense and thus cannot serve as a messenger but requires its own endogenous RNA polymerase for transcription of mRNA. The RNA genome is encapsidated by the nucleocapsid (N) protein in combination with two minor proteins, i.e. RNA-dependent RNA polymerase (L) and phosphoprotein (P) to form the RNP core. The membrane component contains two proteins: an trans-membrane glycoprotein (G) and a matrix (M) protein located at the inner side of the membrane. The G-protein is responsible for cell attachment and membrane fusion in RV, and additionally is the main target for the host immune system.
During transcription, the genome directs the sequential synthesis of a short leader RNA and five monocistronic, capped and polyadenylated mRNAs. During replication, the conditional transcription stop and start signals between the cistrons are ignored by the viral polymerase. For both the transcriptase and the replicase reaction the presence of the N-protein complexed with the RNA genome as well as the L- and P-proteins are required. The gene order on the RV genome has been determined and is 3'-leader-N-P-M-G-L-5' as shown in FIG. 1. Each of the mRNAs of RV is translated immediately after transcription. Two events--occur sequentially during replication: first the production of an encapsidated complete positive strand RNA complementary to the genome, followed by the production of complete negative-stranded RNA which is also encapsidated by the N, L and P proteins. Finally, the newly assembled RNP cores associate with M-protein and G-protein during the assembly and budding process leading to the release of fully formed and infectious RV virions.
The 11.9 kb genomic RV RNA contains five open reading frames (ORFs) coding for the N, P, M, G and L proteins, in addition to the presence of a pseudogene region (.psi.) between the G and L genes (FIG. 1).
Current vaccines for non-segmented negative strand RNA viruses comprise chemically inactivated virus vaccines or modified live virus vaccines comprising an attenuated virus strain the pathogenicity of which is decreased by multiple passages in cell culture. Chemically inactivated rabies vaccines are e.g.: Rabivac, Behringwerke (human), HDC, Rhone-Poulenc (human), Bayovac-LT, Bayer (vet), Madivac, Hoechst (vet), Epivax-LT, Pitman-Moore, Rabisin, Rhone-Merieux. For RV examples of such attenuated viruses are the vaccine strains SAD B19 and ERA. Inactivated vaccines generally induce only a low level of immunity, requiring repeated immunizations. Furthermore, the neutralization inducing antigenic determinants of the pathogens may become altered by the inactivation treatment, decreasing the protective potency of the vaccine.
In general, attenuated live virus vaccines are preferred because they evoke an immune response often based on both humoral and cellular reactions. However, during cell culture passaging uncontrolled mutations may be introduced into the viral genome, resulting in a population of virus particles heterogeneous with regard to virulence and immunizing properties. Over attenuation during passage in cell culture can also be a problem with these vaccines. One must achieve a delicate balance between ensuring that the vaccine is not virulent while making certain that it is still protective. In addition it is well known that such traditional attenuated live virus vaccines can revert to virulence resulting in disease outbreaks in inoculated animals and the possible spread of the pathogen to other animals.
Moreover, a problem with combined live viral vaccines is the mutual influence of the antigenic components resulting in a decrease of the potency of one or more of the constituting components.
Furthermore, with currently administered live attenuated or inactivated RV vaccines it is not possible to determine whether a specific animal is a carrier of RV field virus or whether the animal was vaccinated. Hence, it can be important to be able to discriminate between animals vaccinated with a RV vaccine and those infected with a field virus so as to be able to take appropriate measures to reduce spreading of a virulent field virus. The introduction of for example a serologically identifiable marker can be achieved by introducing a mutation in a gene encoding a (glyco-) protein of RV which normally give rise to the production of antibodies in an infected host animal.
It is desired to introduce a mutation into the RV RNA genome in a controlled manner such that for example the resulting mutant RV is attenuated or comprises a heterologous nucleic acid sequence encoding epitopes of foreign proteins, e.g. immunological marker proteins or antigens of pathogens. Recombinant DNA techniques are already widely used for this purpose with DNA viruses and positive strand RNA viruses. Examples for recombinant DNA viruses: Aujeszky virus (PRV); Adenoviruses; Vaccinia viruses. Examples for recombinant positive-strand RNA viruses: Alphaviruses (Sindbis V., Semliki forest virus: H. V. Huang, C. M. Rice, C. Xiong, S. Schlesinger (1989) RNA viruses as gene expression vectors. Virus Genes 3, 85-91). Picornaviruses (Polio virus, Hepatitis A-virus, Foot- and mouth-disease virus: J. W. Almond and K. L. Burke (1990) Poliovirus as a vector for the presentation of foreign antigens. Semin. Virol. 1, 11-20). Directed genetic manipulation of RNA virus genomes depends on the ability to produce recombinant RNAs which are accepted as a template by the particular RNA-dependent RNA polymerases. Transcripts generated by many standard DNA-dependent RNA polymerases (e.g. T7 RNA polymerase or cellular RNA polymerase II) and mimicking viral genomes are recognized by the polymerases of many positive stranded RNA viruses. This allowed recovery of infectious viruses or replicons from cDNA transcripts and the application of recombinant DNA technology to manipulate these genomes in a site specific manner. Since RNAs corresponding to the genomes of positive stranded RNA viruses may function as mRNA for translation of the viral polymerases, an infectious cycle may be initiated by introduction of the genome analogs into a cell. The template of the polymerases of negative-stranded RNA viruses, however, exclusively is the RNP complex. Moreover, and in contrast to positive stranded RNA viruses, their genomic or antigenomic RNA may not function as mRNA and thus all viral proteins involved in replication and transcription of artificial RNAs have to be provided in trans.
An appropriate system for encapsidation of genomic RNA analogs of a negative-stranded RNA viruses with a segmented genome in order to provide the appropriate template is recently disclosed by Palese, P. et al., (WO 91/03552). RNA transcripts from influenza virus genome segments were encapsidated by purified proteins in vitro which can be used to transfect cells together with a helper virus. However, it was found that this approach was not successful with RV, a virus having a non-segmented genome. Short model genomes of VSV and RV lacking the major part of the RNA genome comprising the genes encoding the viral proteins could be encapsidated and expressed by plasmid encoded proteins (Pattnaik, A. K. et al, Cell 69, 1011-1020, 1992; Conzelmann, K-K. and M. Schnell, J. Virology 68, 713-719, 1994). This approach involved the co-expression of both the genome analogs optionally comprising reporter gene inserts, and particular viral proteins from transfected plasmids in order to produce defective virus particles. Ballart et al. described a method to obtain infectious measles virus, also a non-segmented negative-stranded RNA virus, from cloned cDNA (The EMBO Journal, 9: 379-384 (1990)). A European Patent Application relating to this method was filed with the author as one of the inventors.
Both the paper and the Application were withdrawn however, since further research revealed that all supposed recombinant viruses were no recombinants at all, but mere progeny virus of the originally used vaccine strain.
Thus it must be concluded, that attempts to obtain infectious recombinant negative-stranded RNA viruses with a large, non-segmented genome which necessitates manipulation of the entire genomes, have failed until now.